We have recently discovered that loss of organic anion transporter 3 (OAT3), but not loss of the related renal transporters OAT1 and RST (URAT1), results in decreased blood pressure in mice. This suggests that an endogenous compound(s) specifically transported by OAT3 is involved in the regulation of blood pressure, and our preliminary data indicate that both renal and vascular mechanisms may contribute. We propose in Specific Aim 1 to investigate the potential renal mechanisms. OAT3 is expressed in the renal proximal tubule, distal tubule, and collecting duct, and its endogenous substrates include suppressors of tubular NaCl transport such as PGE2. Moreover, preliminary studies show that the non-selective OAT inhibitor, probenecid, lowers the activity of the epithelial sodium channel, ENaC, in the collecting duct, and Oat3 knockout (-/-) mice manifest significantly greater weight loss in response to a low NaCl diet. These data suggest that OAT3 may contribute to upregulation of renal NaCl reabsorption and thereby to blood pressure regulation. We will perform renal cross-transplantation to differentiate roles of renal versus extra-renal OAT3, and determine differences between Oat3-/- and wild-type (+/+) mice in renal NaCl transport, ENaC activity, and the role of PGE2, so as to identify sites of impaired NaCl reabsorption and/or altered regulation. In Specific Aim 2, we will investigate potential vascular mechanisms underlying blood pressure regulation by OAT3. We previously identified thymidine as a novel endogenous OAT3 substrate that accumulates in the plasma of Oat3-/- mice and acutely lowers blood pressure. We now propose to assess vascular tone in Oat3-/- and +/+ mice, and to determine effects of thymidine and other potentially vasoactive OAT3 substrates, including PGE2 and cGMP. Finally, since loss of OAT3 results in decreased blood pressure, pharmacological inhibition of OAT3 function might also decrease blood pressure, raising the possibility of a novel approach to the design of antihypertensive drugs. Preliminary data show that administration of non-selective OAT inhibitors can reduce blood pressure in mice with both normal and increased blood pressure. We propose in Specific Aim 3 to screen for selective OAT3 inhibitors as a means to identify novel compounds with antihypertensive potential. Specifically, we will first identify OAT3 inhibitors via iterative in silico and in vitro steps (characterize an initial set of inhibitors to generate a pharmacophore; use the pharmacophore to perform in silico screens of large chemical structure databases; test high-scoring matches from these screens for OAT3 inhibition in vitro). We will then test the most potent of the identified inhibitors for antihypertensive capacity in vivo. We provide preliminary data indicating the feasibility of these Aims (including identification of selective OAT3 inhibitors), and have enlisted the support of expert collaborators. The proposed work thus has the potential to significantly increase our understanding of mechanisms of blood pressure regulation and to thereby impact the medical treatment of hypertension.